Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells, we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo, including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy.
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The authors are indebted to M. Karimova (TU Dresden; TUD) for providing reagents, R. Kuhnert (TUD) for help with mouse work, A. Käßner-Frensel (TUD) for SKY analyses, R. Naumann (Max Planck Institute for Molecular Cell Biology and Genetics; MPI-CBG) for the generation of transgenic mice, S. Winkler (MPI-CBG) and A. Dahl (DFG-Forschungszentrum für Regenerative Therapien Dresden) for DNA sequencing, D. Dubrau (Heinrich Pette Institute) for initial ASLV-vector construction, G. Pilnitz-Stolze, B. Weseloh and B. Abel (Heinrich Pette Institute) for technical assistance, and D. Indenbirken and M. Alawi (Heinrich Pette Institute) for next-generation sequencing of nrLAM-PCR products. This work was supported by the DFG (BU 1400/7-1), the BMBF (GO-Bio FKZ 0315090), the Else Kröner-Fresenius-Stiftung (2010_A82), and the “Viral Latency” program at the Heinrich Pette Institute–Leibniz Institute for Experimental Virology, Hamburg. The reporter plasmid pCAGGS-loxP-mCherry-loxP-EGFP, where a floxed mCherry gene was inserted between the CAG enhancer/promoter module and the EGFP gene, was a generous gift from E.M. Tanaka (DFG-Forschungszentrum für Regenerative Therapien Dresden). The mammalian expression vector pCAGGS-IRES-puro was a generous gift from K. Anastassiadis (TUD).
J.K., J.C., J.H. and F.B. are inventors on a patent application regarding Brec1 (EP 14183277).
Integrated supplementary information
(a) loxBTR subsites used for the combinational directed-evolution process to generate Brec1. Sequence differences (compared to the pool of employed related recombinase target sites) in the enzyme bound half sites of the Brec1 target site loxBTR and loxBTR subsites are marked in red or green for the reverse complementary half site. The spacer sequences are shown in grey. (b) Evolution vector pEVO-recomb-target. In E. coli, the recombinase is expressed from the pBAD promoter upon induction with L-arabinose. The vector also contains the regulatory gene araC and a chloramphenicol resistance marker (Cmr). Recombination at the target sites leads to deletion of the intervening region containing a unique NdeI restriction site; following DNA cleavage with NdeI, successful candidates were retrieved by PCR using primers P1 and P3. Primer P2 was employed for PCR amplification of the full-length recombinase coding sequence after DNA shuffling. BsrGI and XbaI restriction sites flanking the recombinase coding sequence facilitate convenient cloning of the recombinases and simple assaying of recombination efficiency in bacteria. The sizes of the unrecombined and recombined (Δ) forms of the vector are indicated.
The NLS sequence, added for nuclear localization of Brec1 in mammalian cells, is shown in blue. Mutations and their corresponding amino acid number in the Cre sequence are shown in red, evolutionary conserved mutations are shown in bold. DNA contact residues based on the Cre crystal structure are underlined.
(a) Schematic representation of recombination of reporter constructs stably integrated in the genome. After recombination, PCR results in a 0.7 kb product (one triangle), while non-recombined template produces a 1.8 kb PCR product (two triangles). (b) Reporter HeLa cell lines with stably integrated loxBTR target sites were transfected with recombinase expression plasmid. Genomic DNA was isolated and assayed for recombination by PCR using primers F and R that anneal to the integrated DNA regions: -ctr, transfection of wild type cells (lane 1); +ctr, transfection with recombined reporter (lane 2); lanes 3-6 show results from cells carrying genomic loxBTR sites transfected with the indicated expression plasmids; M, DNA marker lane. (c) Reporter HeLa cell lines with stably integrated target sites loxBTR, loxP or loxLTR were transfected with recombinase Brec1 expression plasmid (lanes 1-3). Genomic DNA was isolated and assayed for recombination by PCR using primers F and R. Controls for cells harboring the loxP or LoxLTR reporters, transfected with Cre or Tre recombinase expression vectors are shown in lanes 4 and 5.
Exponentially growing Jurkat T cells were transduced with LV-cBrec1, encoding constitutively expressed Brec1 recombinase, with LV-Brec1 (Tat-inducible promotor configuration), or mock transduced, respectively. Transduced GFP+ cells were enriched by FACS. (a) Transduced cells (3 x 106) were seeded into dishes and were grown for 11 days. At indicated time point cells were counted and total cell numbers were calculated. (b) Cell cycle progression monitored by DNA staining at week 6 of Brec1 expression. Jurkat cells were harvested, fixed, and DNA content was stained with propidium iodide. Signals were measured by analytic flow cytometry. (c) Primary human CD4+ T cells were transduced with LV-cBrec1 (constitutive promoter configuration), with LV-Ctr (encoding GFP only), or were mock transduced. Transduced GFP+ cells were enriched by FACS and cultured for two weeks. Subsequently, expression of Brec1 or GAPDH (control) was analyzed by RT-PCR. (d) Analysis of apoptosis in primary human CD4+ T cells at week two post transduction by Annexin V/propidium iodide staining. (e) Functionality of LV-cBrec1 transduced primary CD4+ T cells at two weeks post transduction tested by flow cytometric analysis of CD154 and IFN expression after 3 hours of PMA/ionomycin stimulation. (f) Secretion pattern of Th1-, Th2- and Th17-specific cytokines in primary transduced CD4+ T cells after PMA/Ionomycin stimulation two weeks after transduction as determined by multiplex ELISA. The pattern matches that of non-transduced cells (mock). (g) IL4-specific Elispot analysis of human primary mock or LV-cBrec1 transduced CD4+ T cells after PMA/Ionomycin stimulation two weeks after transduction. (h) CD34+ HSC were mock-transduced or transduced with LV-Ctr, LV-Brec1, or LV-cBrec and 100 cells were seeded into cytokine-containing methyl-cellulose. Culture dishes were incubated for 14 days before counting colonies.
(a) Nucleotide sequences of human genomic sites that most closely resemble the loxBTR sequence and their locations in the human genome. Sequences are aligned to loxBTR. Nucleotides that differ from loxBTR are shown in red. (b) Agarose gel showing the activity of Brec1 on loxBTR (lanes 1 and 5, positive control) and the lack of activity for the six loxBTR-like human genomic sites HGS1 – HGS6 (lanes 2-4 and 6-8). E. coli cells harboring the pEVO vector containing the Brec1 coding sequence and indicated target sites were grown at 1 mg/ml L-arabinose. Recombination was assayed by restriction enzyme digest, resulting in a smaller fragment for recombined (one triangle) and a larger fragment for non-recombined substrate (two triangles). M, DNA marker. (c) SKY (spectral karyotyping) analysis of metaphase spreads isolated from primary human CD4+ T cells constitutively expressing Brec1 (LV-cBrec1). An RGB display of the 24-colour SKY hybridization of a representative normal metaphase is shown. Number of analyzed chromosome spreads: 27.
(a) HeLa-smurf cells, containing at the PACS1 locus on chromosome 11 a full-length HIV-1 proviral DNA that expresses blue fluorescent protein (BFP), have been previously described29 At a distance of ~ 1 Mb, an additional self-contained reporter construct was inserted at the KDMA2 locus via CRISPR/Cas9 technology. This reporter simultaneously expresses from an HIV-1 LTR promoter the fluorescent protein mKO2 and the HIV trans-activator Tat by use of an ERAV 2A-like sequence. (b) Brec1-mediated provirus recombination results in an excision product that can be detected by PCR (depicted in Fig. 4c). At day 3 post transduction with LV-Brec1, this circular excision product was observed in chromosomal DNA isolated from HeLa-smurf reporter cells. Brec1, LV-Brec1 transduced cells; NC, negative control (non-transduced cells); PC, positive PCR control; H2O, negative PCR control (omitting DNA template). (c) Analysis of potential aberrant Brec1-mediated loxBTR recombination. To monitor LTR/genomic DNA junctions, and LTR/proviral genome junctions, non-restrictive linear amplification-mediated PCR (nrLAM-PCR) was performed as described previously29, using an mKO2-specific anchor primer (indicated in panel a). In addition to extension products from the reporter construct integrated at the KDM2A locus (product I in panel a), aberrant recombination between distant loxBTR sequences would be expected to yield nrLAM extension products which contain either proviral or 5’-proximal host sequences from the PACS1 locus (products II and III in panel a, respectively). As shown in panel c, PCR analysis with primer pairs designed to amplify upstream integration site sequences from extension products I and III (indicated in panel a) revealed the expected amplification product (asterisk) for primer pair P1/P2, but not for primer pair P3/4, indicating that aberrant recombination (i.e. recombination of the proviral 5’LTR and the LTR of the reporter construct) did not occur. M, DNA marker; PC positive PCR control; mock, nrLAM-PCR template from non-transduced cells; Brec1, nrLAM-PCR template from LV-Brec1 transduced cells; H2O, negative PCR control (omitting DNA template). Additionally, next generation bulk sequencing of nrLAM amplicons (2x250 bp paired end, Illumina MiSeq platform) yielded 1698 reads with 5’-fragments that spanned the host/loxBTR junction of the reporter construct but none that would indicate aberrant recombination, again indicating that Brec1 expression resulted exclusively in provirus excision.
(a) Schematic representation of the Brec1 expression cassette. Brec1 is expressed from the cytomegalovirus (CMV) immediate early enhancer-chicken beta-actin hybrid (CAG) promoter. Breeding strategy following generation of Brec1 founder mice by pronuclear injection: WT, wild type mouse; TGM, transgenic mouse. The number of offspring and the TG carriers indicated for the first and second generation show that the transgene is inherited with a Mendelian ratio. (b) Agarose gel showing PCR-based genotyping of one founder (lane 2) and three transgenic offspring (lanes 3-5) using Brec1-specific primers. WT, wild type control (lane 1); M, DNA marker lane. (c) Agarose gel showing PCR-based analysis of cDNA generated by reverse transcription of mRNA isolated from WT animal (control, lane 1) and three transgenic Brec1 mice (TGM1-TGM3, lanes 2-7): +, reverse transcriptase added to RT reaction; -, no-reverse transcriptase control. (d) Schematic representation of the lentiviral reporter construct. Important elements are indicated. The vector backbone has been previously described in detail29 (e) Schematic drawing of the primer binding sites within the lentiviral reporter construct. Important elements are indicated and the size of the expected amplicon for primers P3 and P4 is shown. Brec1-mediated recombination leads to the formation of a circular excision product that can be detected employing primer P1 and P2. (f) Schematic of the 600 bp amplification product for primers P1 and P2. A sequencing read is shown confirming the expected sequence. The loxBTR sequence is marked. (g) Confirmation of lentiviral integration of the reporter construct. PCR assays for indicated animals is provided. The 1.7 kb fragment resulting from amplification with primers P3 and P4 is marked by an asterisk. (h) Confirmation of Brec-1 mediated recombination. Gel showing PCR analysis using DNA isolated from cells of indicated animals. The product showing the recombined band is indicated with P1/P2.
Supplementary Figure 8 Analysis of Brec1 activity in mice engrafted with CD4+ T cells isolated from HIV infected patients.
(a) Monitoring of viral load (red dots) and percentage of human CD45+CD4+ cells (blue dots) in hu-Rag2 mice engrafted with negative control vector (LV-Ctr) transduced primary CD4+ T cells isolated from PBMC of HIV infected patients. (b) Antiviral activity and host cell protection in hu-Rag2 mice engrafted with LV-Brec1 transduced patient-derived CD4+ T lymphocytes. Human cells in PBMC, spleen and liver of the respective Brec1-expressing animal was determined at necropsy by FACS using single cell suspensions (bar chart).
Supplementary Figures 1–8 and Supplementary Table 3 (PDF 8079 kb)
Representation of recombinase target sites loxP, loxLTR, and loxBTR in HIV-1 LTR sequences of the most prevalent subtypes (XLS 27 kb)
Conserved amino acids emerging during substrate-linked evolution (XLS 30 kb)
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Karpinski, J., Hauber, I., Chemnitz, J. et al. Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity. Nat Biotechnol 34, 401–409 (2016) doi:10.1038/nbt.3467
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